The present invention relates to gene therapy for the treatment of tumors. The invention more particularly relates to introduction of a gene encoding an anti-angiogenic factor into cells of a tumor, for example with an adenovirus vector, to inhibit growth or metastasis, or both, of the tumor.
Cell migration is a coordinated process that bridges cellular activation and adhesion whereas the equilibrium between pericellular proteolysis and its inhibition (e.g., triggered by plasminogen activator inhibitors and tissue inhibitors of metalloproteinases) is disrupted (1-3). Urokinase plasminogen activator (uPA) is a pivotal player in this process because it initiates a proteolytic cascade at the surface of migrating cells by binding to its cell surface receptor (uPAR) (4, 5). Binding of uPA to its receptor greatly potentiates plasminogen/plasmin conversion at the cell surface (6). Plasmin is a broadly specific serine protease which can directly degrade components of the extracellular matrix such as fibronectin, vitronectin or laminin. Plasmin also indirectly promotes a localized degradation of the stroma by converting inactive zymogens into active metalloproteinases (7). The selective distribution of uPAR at the leading edge of migrating cells (invadopodes) apparently concentrates uPA secreted by themselves or by neighboring stroma cells (8). uPAR is also directly involved in cellular adhesion to the extracellular matrix as illustrated by its uPA-dependent binding to vitronectin (9), and because uPAR modulates the binding properties of several integrin molecules (10). Finally, uPA and plasmin are somehow involved in cell morphogenesis by activating or inducing the release of morphogenic factors such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), fibroblast growth factors (FGFs) and transforming growth factor xcex2 (TGFxcex2) (11, 12).
Taken together, these observations indicate that the uPA/uPAR system controls cell migration by coordinating cellular activation, adhesion and motility. This statement is supported by clinical observations that correlate the presence of enhanced uPA activity at the invasive edge of the tumors (13, 14). That melanoma induced by DMBA and croton oil do not progress to a malignant stage in uPA-deficient mice also support a role of uPA in tumor establishment and progression (15).
uPA binds to uPAR by its light chain fragment, also known as amino-terminal fragment (ATF, amino acid 1-135). This interaction is species restricted (16) and involves the EGF-like domain of ATF (residues 1-46), in which amino acid 19-32, which are not conserved between mice and human, are critical (17, 18). ATF-mediated disruption of the uPA/uPAR complex inhibits tumor cell migration and invasion in vitro (19). Intraperitoneal bolus injection of a chimeric human ATF-based antagonist has also been used to inhibit lung metastases of human tumor cells implanted within athymic mice, without significantly affecting primary tumor growth (20). A further study reported that intraperitoneal injection of synthetic peptides derived from murine ATF was effective in inhibiting both primary tumor growth and lung metastases (21). These results are consistent with a role of the uPA/uPAR complex in controlling the motility of both tumor and endothelial cells (22). That a chimeric murine ATF-based antagonist could inhibit vessel growth in an artificial bFGF-enriched extracellular matrix (23) further supports uPA/uPAR involvement in controlling angiogenesis in vivo.
The formation of blood vessels, or angiogenesis, results from the capillary growth of pre-existing vessels. Angiogenesis is essential for a number of physiological processes such as embryonic development, wound healing and tissue or organ regeneration. Abnormal growth of new blood vessels occurs in pathological conditions such as diabetic retinopathy and tumor growth, as well as tumor dissemination to distant sites [38,24]. Both experimental and clinical studies have showed that primary tumors as well as metastasis remain dormant due to a balanced rate of proliferation and apoptosis unless the angiogenesis process is switched on [39].
The growth of endothelial cells is tightly regulated by both positive and negative factors. The onset of tumor angiogenesis could be triggered either by an upregulation of tumor-released angiogenic factors such as vascular endothelial growth factor (VEGF) and acid or/and basic fibroblast growth factor (bFGFs), or by a downregulation of angiostatic factors such as thrombospondin and angiostatin [39]. Both the reconstitution of angiostatic factors and the removal of angiogenic stimulating factors thus constitute plausible clinical strategies to suppress tumor angiogenesis [40, 41]. Angiostatic-based therapies should also apply to all solid tumors because endothelial cells do not vary from one tumor type to the other, further emphasizing the clinical relevance of such an anti-cancer approach. Thus, the therapy targeting angiogenesis appears to be highly relevant to clinical practice.
Many physiological angiostatic factors are derived upon proteolytic cleavage of circulating proteins. This is the case for angiostatin [32], endostatin [42], the 16 kDa fragment of prolactin [43], or platelet factor-4 [44]. Angiostatin was initially isolated from mice bearing a Lewis lung carcinoma (LLC), and was identified as a 38 kDa internal fragment of plasminogen (Plg) (aa 98-440) that encompasses the first four kringles of the molecule [32; WO95/29242; U.S. Pat. No. 5,639,725]. Angiostatin has been shown to be generated following hydrolysis of Plg by a metalloelastase from GM-CSF-stimulated tumor-infiltrating macrophages [45]. Intraperitoneal bolus injections of purified angiostatin in six different tumor models have proved to be very effective in suppressing primary tumor growth, with no apparent toxicity [46]. Angiostatin-mediated suppression of tumor angiogenesis apparently drove the tumor cells into a higher apoptotic rate that counterbalanced their proliferation rate. In this study, tumor growth usually resumed following removal of the angiostatin molecule, emphasizing the importance of achieving long-term delivery for optimal clinical benefits [46]. In vitro studies with recombinant proteins indicated that the angiostatic activity of angiostatin was mostly mediated by kringles 1-3, thus leaving a minor activity for kringle 4 [47]. As for most angiostatic factors, little is known about the molecular pathway by which angiostatin exerts its effect.
As angiostatic therapy will require a prolonged maintenance of therapeutic levels in vivo, the continuous delivery of a recombinant protein will be expensive and cumbersome. Direct in vivo delivery of the corresponding genes with viral vectors constitutes an attractive solution to this problem. Because most cancer gene therapies currently rely on destructive strategies that target the tumor cells [48], viral-mediated gene delivery of an angiostatic factor represents a conceptually different, and possibly synergistic, approach to fight cancer.
Despite these results, there remains a need to develop effective treatments for tumors, particularly chemotherapy-resistant tumors.
The present invention addresses this need by establishing an effective mode for treating a tumor.
Various references are cited in this specification by number, which are fully set forth after the Examples. None of the references cited herein should be construed as describing or suggesting the invention disclosed herein.
The present invention advantageously provides a highly effective gene therapy for tumors. Indeed, in a specific embodiment of the invention murine urokinase ATF expressed by human tumor cells in an athymic murine model unexpectedly effectively inhibits tumorigenicity. In another embodiment, angiostatin expressed in tumor cells in a murine model inhibited tumor growth and tumorigenesis, and induced tumor cell apoptosis, in addition to blocking angiogenesis.
In a broad aspect, the present invention provides a method for inhibiting growth or metastasis, or both, of a tumor comprising introducing a vector comprising a gene encoding an anti-angiogenic factor operably associated with an expression control sequence that provides for expression of the anti-angiogenic factor into a cell or cells of the tumor. Preferably, the vector is a virus vector; more preferably the virus vector is an adenovirus vector. In a specific embodiment exemplified infra, the adenovirus vector is a defective adenovirus vector.
The methods of the invention are useful in the treatment of many tumors, as set forth in detail herein. For example, in specific embodiments, the tumor is a lung carcinoma or a breast carcinoma.
In addition, the invention demonstrates for the first time the advantages of expression of an anti-angiogenic factor by the transduced tumor cells. Accordingly, a gene encoding any anti-angiogenic factor, such as a soluble receptor for an angiogenic protein, or an angiogenesis antagonist, can be delivered in the practice of the invention. In a specific embodiment, the anti-angiogenic factor comprises a sequence of an amino terminal fragment of urokinase having an EGF-like domain, with the proviso that the factor is not urokinase. For example, the anti-angiogenic factor may be a chimeric protein comprising ATF-immunoglobulin or ATF-human serum albumin. In a preferred embodiment, exemplified infra, the anti-angiogenic factor is an amino terminal fragment of urokinase having an amino acid sequence of urokinase from about amino acid residue 1 to about residue 135. In a specific aspect, the urokinase is murine urokinase. In a more preferred aspect, the urokinase is human urokinase.
In an alternative embodiment, the anti-angiogenic factor is angiostatin, in particular, kringles 1 to 3 of angiostatin. In a particularly preferred embodiment, the anti-angiogenic factor is the amino-terminal fragment of plasminogen (Plg) having an amino acid sequence of plasminogen from about amino acid residue 1 to about residue 333. In another preferred embodiment, the anti-angiogenic factor is the amino-terminal fragment (angiostatin) from human plasminogen.
In a related embodiment, the invention is directed to use of a vector comprising a gene encoding an anti-angiogenic factor operably associated with an expression control sequence that provides for expression of the anti-angiogenic factor in the manufacture of a medicament for inhibiting growth or metastasis, or both, of a tumor. More particularly, the invention provides for use of a virus vector of the invention, e.g., as set out below, in the manufacture of a medicament for inhibiting growth or metastasis, or both, of a tumor.
Naturally, in addition to the foregoing methods and uses, the invention provides a novel virus vector comprising a gene encoding an anti-angiogenic factor operably associated with an expression control sequence. In a preferred embodiment, the virus vector is an adenovirus vector. In a more preferred embodiment, the virus vector is a defective adenovirus vector. The virus vectors of the invention can provide a gene encoding any anti-angiogenic factor, as set forth above. For example, the anti-angiogenic factor may comprise a sequence of an amino terminal fragment of urokinase having an EGF-like domain, with the proviso that the factor is not urokinase. In a preferred embodiment, the anti-angiogenic factor is an amino terminal fragment of urokinase having an amino acid sequence of urokinase from amino acid residue 1 to about residue 135. In this embodiment, the urokinase may be murine urokinase or, preferably, human urokinase.
The invention further provides a pharmaceutical composition any of the virus vectors of the invention and a pharmaceutically acceptable carrier.
Thus, one object of the invention is to provide gene therapy by delivery of anti-angiogenic factors for treating tumors.
Another object of the invention is to provide a viral vector for delivery of an anti-tumorigenic factor.
Still another object of the invention is to provide an amino terminal fragment of urokinase (ATF) by gene therapy for treatment of a tumor.
Further, another object of the invention is to provide angiostatin by gene therapy for treatment of a tumor.
Yet another object of the invention is to provide angiostatin, particularly kringles 1 to 3 of angiostatin, by gene therapy for treatment of a tumor.